4OU9: Crystal Structure Of Apocarotenoid Oxygenase In The Presence Of Triton X-100

Citation:
Abstract
Carotenoid cleavage enzymes (CCEs) constitute a group of evolutionarily related proteins that metabolize a variety of carotenoid and non-carotenoid substrates. Typically, these enzymes utilize a non-heme iron center to oxidatively cleave a carbon-carbon double bond of a carotenoid substrate. Some members also isomerize specific double bonds in their substrates to yield cis-apocarotenoid products. The apocarotenoid oxygenase from Synechocystis has been hypothesized to represent one such member of this latter category of CCEs. Here, we developed a novel expression and purification protocol that enabled production of soluble, native ACO in quantities sufficient for high resolution structural and spectroscopic investigation of its catalytic mechanism. High performance liquid chromatography and Raman spectroscopy revealed that ACO exclusively formed all-trans products. We also found that linear polyoxyethylene detergents previously used for ACO crystallization strongly inhibited the apocarotenoid oxygenase activity of the enzyme. We crystallized the native enzyme in the absence of apocarotenoid substrate and found electron density in the active site that was similar in appearance to the density previously attributed to a di-cis-apocarotenoid intermediate. Our results clearly demonstrated that ACO is in fact a non-isomerizing member of the CCE family. These results indicate that careful selection of detergent is critical for the success of structural studies aimed at elucidating structures of CCE-carotenoid/retinoid complexes.
PDB ID: 4OU9Download
MMDB ID: 118500
PDB Deposition Date: 2014/2/15
Updated in MMDB: 2014/05
Experimental Method:
x-ray diffraction
Resolution: 2  Å
Source Organism:
Similar Structures:
Biological Unit for 4OU9: monomeric; determined by author and by software (PISA)
Molecular Components in 4OU9
Label Count Molecule
Protein (1 molecule)
1
Apocarotenoid-15,15'-oxygenase
Molecule annotation
Chemicals (2 molecules)
1
1
2
1
* Click molecule labels to explore molecular sequence information.

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